This application is a 371 of PCT/SE00/00052 filed Jan. 13, 2000.
This invention relates to novel pharmaceutically useful compounds, in particular compounds that are, or are prodrugs of, competitive inhibitors of trypsin-like serine proteases, especially thrombin, their use as medicaments, pharmaceutical compositions containing them and synthetic routes to their production.
Blood coagulation is the key process involved in both haemostasis (i.e. the prevention of blood loss from a damaged vessel) and thrombosis (i.e. the formation of a blood clot in a blood vessel, sometimes leading to vessel obstruction).
Coagulation is the result of a complex series of enzymatic reactions. One of the ultimate steps in this series of reactions is the conversion of the proenzyme prothrombin to the active enzyme thrombin.
Thrombin is known to play a central role in coagulation. It activates platelets, leading to platelet aggregation, converts fibrinogen into fibrin monomers, which polymerise spontaneously into fibrin polymers, and activates factor XIII, which in turn crosslinks the polymers to form insoluble fibrin. Furthermore, thrombin activates factor V and factor VIII leading to a xe2x80x9cpositive feedbackxe2x80x9d generation of thrombin from prothrombin.
By inhibiting the aggregation of platelets and the formation and crosslinking of fibrin, effective inhibitors of thrombin would be expected to exhibit antithrombotic activity. In addition, antithrombotic activity would be expected to be enhanced by effective inhibition of the positive feedback mechanism.
Further, it is known that administration of prodrugs of thrombin inhibitors may give rise to improvements m:
(a) certain pharmacokinetic properties after administration of; and
(b) the prevalence of certain side effects associated with, those inhibitors.
The early development of low molecular weight inhibitors of thrombin has been described by Claesson in Blood Coagul. Fibrinol. (1994) 5, 411.
Blombxc3xa4ck et al (in J. Clin. Lab. Invest. 24, suppl. 107, 59, (1969)) reported thrombin inhibitors based on the amino acid sequence situated around the cleavage site for the fibrinogen Aa chain. Of the amino acid sequences discussed, these authors suggested the tripeptide sequence Phe-Val-Arg (P9-P2-P1, hereinafter referred to as the P3-P2-P1 sequence) would be the most effective inhibitor.
Thrombin inhibitors based on dipeptidyl derivatives with an xcex1,xcfx89-aminoalkyl guanidine in the P1-position are known from U.S. Pat. No. 4,346,078 and International Patent Application WO 93/11152. Similar, structurally related, dipeptidyl derivatives have also been reported. For example International Patent Application WO 94/29336 discloses compounds with, for example, aminomethyl benzamidines, cyclic aminoalkyl amidines and cyclic aminoalkyl guanidines in the P1-position (International Patent Application WO 97/23499 discloses prodrugs of certain of these compounds); European Patent Application 0 648 780, discloses compounds with, for example, cyclic aminoalkyl guanidines in the P1-position.
Thrombin inhibitors based on peptidyl derivatives, also having cyclic aminoalkyl guanidines (e.g. either 3- or 4-aminomethyl-1-amidino-piperidine) in the P1-position are known from European Patent Applications 0 468 231, 0 559 046 and 0 641 779.
Thrombin inhibitors based on tripeptidyl derivatives with arginine aldehyde in the P1-position were first disclosed in European Patent Application 0 185 390.
More recently, arginine aldehyde-based peptidyl derivatives, modified in the P3-position, have been reported. For example, International Patent Application WO 93/18060 discloses hydroxy acids, European Patent Application 0 526 877 des-amino acids, and European Patent Application 0 542 525 O-methyl mandelic acids in the P3-position.
Inhibitors of serine proteases (e.g. thrombin) based on electrophilic ketones in the P1-position are also known. For example, European Patent Application 0 195 212 discloses peptidyl xcex1-keto esters and amides, European Patent Application 0 362 002 fluoroalkylamide ketones, European Patent Application 0 364 344 xcex1,xcex2,xcex4-triketocompounds, and European Patent Application 0 530 167 xcex1-alkoxy ketone derivatives of arginine in the P1-position.
Other, structurally different, inhibitors of trypsin-like serine proteases based on C-terminal boronic acid derivatives of arginine and isothiouronium analogues thereof are known from European Patent Application 0 293 881.
More recently, thrombin inhibitors based on peptidyl derivatives have been disclosed in European Patent Application 0 669 317 and International Patent Applications WO 95/35309, WO 95/23609, WO 96/25426, WO 97/02284, WO 97/46577, WO 96/32110, WO 96/31504, WO 96/03374, WO 98/06740 and WO 97/49404.
However, there remains a need for effective inhibitors of trypsin-like serine proteases, such as thrombin. There is also a need for compounds which are both orally bioavailable and selective in inhibiting thrombin over other serine proteases, in particular those involved in haemostatis. Compounds which exhibit competitive inhibitory activity towards thrombin would be expected to be especially useful as anticoagulants and therefore in the therapeutic treatment of thrombosis and related disorders.
According to the invention there is provided a compound of formula I, 
wherein
R1 represents a N(R5)R6 or a S(O)mR7 substituent;
R2 and R3 independently represent an optional substituent selected from halo, C1-4 alkyl or C1-4 alkoxy (which latter two groups are optionally substituted by halo);
Y represents C1-3 alkylene, optionally substituted by C1-4 alkyl, methylene, xe2x95x90O or hydroxy);
R4 represents H, OH, OR8a, C(O)OR8b or R8c;
R5 represents C1-6 alkyl (optionally substituted by halo) or, together with
R6 and the nitrogen atom to which R5 and R6 are attached, represents a 3- to 7-membered nitrogen containing ring, which ring optionally includes an oxygen atom and/or is optionally substituted with a xe2x95x90O group;
R6 represents C1-6 alkyl (optionally substituted by halo), C(O)R9 or, together with R5 and the nitrogen atom to which R5 and R6 are attached, represents a 3- to 7-membered nitrogen-containing ring, which ring optionally includes an oxygen atom and/or is optionally substituted with a xe2x95x90O group;
or the group N(R5)R6 represents the structural fragment Ia, 
R6a represents one or more optional substituents selected from halo, C1-4 alkyl and C1-4 alkoxy (which latter two groups are optionally substituted by halo);
X represents CH or N;
m represents 0, 1 or 2;
R7 represents H, NH2 or C1-6 alkyl;
R8a and R8b independently represent C1-10 alkyl, C1-3 alkylphenyl or C6-10 aryl, or R8a represents C(R10a)(R10b)OC(O)R11, C(R10a)(R10b)N(H)C(O)OR12 or C(R10a)(R10b)OC(O)N(H)R12;
R8c represents C(R10a)(R10b)OC(O)R11, C(R10a)(R10b)N(H)C(O)OR12 or C(R10a)(R10b)OC(O)N(H)R12;
R10a and R10b independently represent, at each occurrence, H or C1-4 alkyl;
R11 represents, at each occurrence, C6-10 aryl, OR12 or C1-7 alkyl (which latter group is optionally substituted by a substituent selected from OH, CO2H and C6-10 aryl);
R12 represents, at each occurrence, C6-10 aryl or C1-6 alkyl (which latter group is optionally substituted by a substituent selected from OH, CO2H and C6-10 aryl);
R9 represents C6-8, alkyl, Het1, C6-10 aryl or C1-4 alkyl substituted by C6-10 aryl; and
Het1 represents a 4- to 12-membered heterocyclic ring, which ring contains one or more heteroatoms selected from oxygen, nitrogen and/or sulfur, and which ring may be fully saturated, partially saturated or aromatic and/or optionally monocyclic, bicyclic and/or benzo-fused; wherein each aryl/phenyl group and Het1 group identified above is optionally substituted by one or more halo, C1-4 alkyl and/or C1-4 alkoxy groups (which latter two groups are themselves optionally substituted by one or more halo groups);
or a pharmaceutically acceptable salt thereof, provided that:
(a) when m represents 1 or 2, then R7 does not represent H; and
(b) when m represents 0, then R7 does not represent NH2; which compounds are referred to hereinafter as xe2x80x9cthe compounds of the inventionxe2x80x9d.
Pharmaceutically acceptable salts include inorganic acid (e.g. hydrogen halide), and organic acid (e.g. acetic, methanesulfonic or trifluoroacetic acid), addition salts.
The compounds of the invention may exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention. Particular tautomeric forms that may be mentioned include those connected with the position of the double bond in the amidine functionality in the compound of formula I, and the position of the substituent R4, when this does not represent H.
The compounds of formula I also contain at least two asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. All diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation, or by derivatisation, for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means (e.g. HPLC, chromatography over silica). All stereoisomers are included within the scope of the invention.
As used herein, the term xe2x80x9carylxe2x80x9d includes phenyl, naphthyl and the like. Alkyl groups which R2, R3, R5, R6, R6a, R7, R8a, R8b, R9, R10a, R10b, R11, and R12 may represent, and with which Y and aryl/phenyl and Het1 groups may be substituted; alkoxy groups which R2, R3 and R6a may represent, and with which aryl/phenyl and Het1 groups may be substituted; the alkyl part of alkylphenyl or alkylaryl groups which R8a, R8b, R9, R11 and R12 may represent; and alkylene groups which Y may represent, may, when there is a sufficient number of carbon atoms, be linear or branched, be saturated or unsaturated, be cyclic, acyclic or part cyclic/acyclic, and/or be optionally interrupted by an O atom. The skilled person will appreciate that when alkyl groups that R2, R3, R5, R6, R6a, R7, R8a, R8b, R9, R10a, R10b, R11 and R12 may represent, and with which Y and aryl/phenyl and Het1 groups may be substituted are cyclic and interrupted by oxygen, they may then represent oxygen-containing heterocycles such as tetrahydrofuranyl or (where appropriate) tetrahydropyranyl.
Halo groups which R2, R3 and R6a may represent, and with which R2, R3, R5, R6, R6a and aryl/phenyl and Het1 groups may be substituted, include fluoro, chloro, bromo and iodo.
Abbreviations are listed at the end of this specification.
When R5 and R6, together with the nitrogen atom to which they are attached, represent a 3- to 7-membered nitrogen-containing (e.g. pyrrolidine) ring, which ring optionally includes an oxygen atom and/or is substituted by a xe2x95x90O group, the ring is preferably substituted at a carbon atom that is xcex1 to the nitrogen atom. For the avoidance of doubt, the nitrogen atom to which R5 and R6 are attached is the nitrogen atom that must be present in the ring.
Compounds of the invention which may be mentioned include those in which:
R2 and R3 independently represent an optional substituent selected from halo or C1-4 alkyl (optionally substituted by halo);
R5 represents C1-6 alkyl or, together with R6 and the nitrogen atom to which R5 and R6 are attached, represents a 3- to 7-membered nitrogen containing ring, optionally substituted with a xe2x95x90O group;
R6 represents C1-6 alkyl, C(O)R9 or, together with R5 and the nitrogen atom to which R5 and R6 are attached, represents a 3- to 7-membered nitrogen-containing ring, optionally substituted with a xe2x95x90O group;
when R4 represents OR8a or C(O)OR8b, R8a and R8b independently represent, at each occurrence, C1-10 alkyl, C1-3 alkylphenyl or C6-10 aryl, which latter two groups are optionally substituted by one or more halo, C1-4 alkyl and/or C1-4 alkoxy groups;
R9 represents C1-6 alkyl; and
all other substituents are otherwise as defined hereinbefore.
Further compounds of the invention that may be mentioned include those in which R4 does not represent R8c.
Preferred compounds of the invention include those in which:
R2, if present, represents linear or branched C1-4 alkyl or C1-4 alkoxy (both of which are optionally substituted by halo), or halo (e.g. chloro);
R3 is either absent or, if present, represents linear or branched C1-4 alkyl, or halo;
R5 represents linear, branched or cyclic C1-6 alkyl or, together with R6 and the nitrogen atom to which R5 and R6 are attached, represents a 4- to 6-membered nitrogen containing ring, optionally substituted with a xe2x95x90O group;
R6 represents linear, branched or cyclic C1-6 alkyl, C(O)xe2x80x94C1-6 alkyl or, together with R5 and the nitrogen atom to which R5 and R6 are attached, represents a 4- to 6-membered nitrogen-containing ring, optionally substituted with a xe2x95x90O group;
R7 represents linear, branched or cyclic C1-6 alkyl;
Y represents CH2 or (CH2)2.
When R4 represents OR8a, preferred compounds of the invention include those in which R8a represents linear or branched C1-6 alkyl, C4-5 cyclic alkyl (which latter two groups are optionally interrupted by oxygen), or phenyl or C1-2 alkylphenyl (e.g. benzyl) (which latter two groups are optionally substituted as specified hereinbefore), or R8a represents CH2OC(O)R11, in which R11 represents phenyl, linear, branched or cyclic C1-6 alkyl (which latter group is optionally substituted by a substituent selected from OH, CO2H and phenyl), or OR12 (wherein R12 represents phenyl or linear, branched or cyclic C1-6 alkyl (which latter group is optionally substituted by a substituent selected from OH, CO2H and phenyl)).
When R4 represents C(O)OR8b, preferred compounds of the invention include those in which R8b represents linear or branched C1-2 alkylphenyl or phenyl (which latter two groups are optionally substituted as specified hereinbefore).
Preferred compounds of the invention include those in which R1 is attached to the phenyl ring at the 3-position, relative to the xe2x80x94CH(OH)xe2x80x94 group to which the phenyl ring is also attached. The optional substituent R2 is preferably attached to the phenyl ring at the 5-position, relative to the xe2x80x94CH(OH)xe2x80x94 group to which the phenyl ring is also attached.
When the group N(R5)R6 represents a structural fragment Ia, the fragment is preferably unsubstituted.
More preferred compounds of the invention include those in which:
R1 represents N(R5)R6;
R3 is either absent or, if present, represents methyl or chloro, preferably in the 2-position relative to the xe2x80x94CH2xe2x80x94 group to which the phenyl ring is also attached;
R8a represents linear or branched C1-4 alkyl (optionally interrupted by oxygen) or C4-5 cyclic alkyl interrupted by oxygen;
R5 represents C1-4 alkyl or, together with R6 and the nitrogen atom to which R5 and R6 are attached, represents a 5- or 6-membered nitrogen containing ring, optionally substituted with a xe2x95x90O group;
R6 represents C1-4 alkyl, C(O)xe2x80x94C1-6 alkyl (e.g. C(O)xe2x80x94C1-4 alkyl) or, together with R5 and the nitrogen atom to which R5 and R6 are attached, represents a 5- or 6-membered nitrogen-containing ring, optionally substituted with a xe2x95x90O group.
Compounds of formula I in which the fragment 
is in the S-configuration are preferred.
Compounds of formula I in which the fragment 
is in the R-configuration are preferred.
The wavy lines on the bonds in the above two fragments signify the bond positions of the fragments.
Preferred compounds of formula I include the compounds of the Examples described hereinafter.
According to the invention there is also provided a process for the preparation of compounds of formula I which comprises:
(i) the coupling of a compound of formula II, 
wherein R1 and R2 are as hereinbefore defined with a compound of formula III, 
wherein Y, R3 and R4 are as hereinbefore defined, for example in the presence of a coupling agent (e.g. EDC, DCC, HBTU, HATU, TBTU, PyBOP or oxalyl chloride in DMF), an appropriate base (e.g. pyridine, 2,4,6-trimethylpyridine, 2,4,6-collidine, DMAP, TEA or DIPEA) and a suitable organic solvent (e.g. dichloromethane, acetonitrile or DMF);
(ii) the coupling of a compound of formula IV, 
wherein R1, R2 and Y are as hereinbefore defined with a compound of formula V, 
wherein R3 and R4 are as hereinbefore defined, for example in the presence of a coupling agent (e.g. oxalyl chloride in DMF, EDC, DCC, HBTU, HATU, PyBOP or TBTU), an appropriate base (e.g. pyridine, 2,4,6-trimethylpyridine, DMAP, TEA, 2,4,6-collidine or DIPEA) and a suitable organic solvent (e.g. dichloromethane, acetonitrile or DMF);
(iii) for compounds of formula I in which R4 represents OH or OR8a, reaction of a compound of formula VI, 
wherein R1, R2, Y and R3 are as hereinbefore defined with a compound of formula VII,
H2NORaxe2x80x83xe2x80x83VII
wherein R1 represents H or R8a and R8b is as hereinbefore defined, for example at between 40 and 60xc2x0 C., in the presence of a suitable base (e.g. TEA) and an appropriate organic solvent (e.g. THF, CH3CN, DMF or DMSO), optionally by pre-treating the compound of formula VI with gaseous HCl, in the presence of a lower alkyl (e.g. C1-6 alkyl) alcohol (e.g. ethanol) at, for example, 0xc2x0 C., to form a compound of formula VIII, 
wherein Rc represents lower (e.g. C1-6) alkyl, such as ethyl, and R1, R2, Y and R3 are as hereinbefore defined, which compound may be isolated if desired;
(iv) for compounds of formula I in which R4 represent OH or OR8a, reaction of a compound corresponding to a compound of formula I, in which, in place of R4, a protecting group C(O)ORb1 is present, in which Rb1 represents a group such as 2-trimethylsilylethyl, C1-6 alkyl or alkylphenyl (e.g. benzyl), with a compound of formula VII as hereinbefore defined, for example under similar reaction conditions to those described hereinbefore for preparation of compounds of formula I (step (iii)) (the skilled person will appreciate that in such a reaction the diprotected amidine (i.e. C(O)ORb1 and ORa protected) derivative may, in some cases, be isolated if desired, and the C(O)ORb1 group then removed using conventional techniques);
(v) for compounds of formula I in which R4 represents C(O)OR8b, reaction of a compound of formula I in which R4 represents H with a compound of formula IX,
L1xe2x80x94C(O)OR8bxe2x80x83xe2x80x83IX
wherein L1 represents a suitable leaving group, such as halo or p-nitrophenoxy, and R8b is as hereinbefore defined, for example 0xc2x0 C. in the presence of a suitable base (e.g. NaOH) and an appropriate organic solvent (e.g. THF) and/or water;
(vi) for compounds of formula I in which R4 represents OR8a, reaction of a corresponding compound of formula I in which R4 represents OH with a compound of formula IXA,
L1xe2x80x94R8axe2x80x83xe2x80x83IXA
wherein R8a and L1 are as hereinbefore defined, for example at between 0xc2x0 C. and reflux temperature, optionally in the presence of an appropriate solvent (e.g. DCM, THF, MeCN or DMF) and a suitable base (e.g. Et3N or pyridine);
(vii) for compounds of formula I in which R4 represents R8c, wherein R8c represents C(R10a)(R10b)OC(O)R11 or C(R10a)(R10b)OC(O)N(H)R12, reaction of a corresponding compound of formula IXB, 
wherein R1, R2, Y, R3, R10a and R10b are as hereinbefore defined with a compound of formula IXC,
L1C(O)R13xe2x80x83xe2x80x83IXC
wherein R13 represents R11 or N(H)R12, and L1, R11 and R12 are as hereinbefore defined, for example under conditions described hereinbefore (process step (vi));
(viii) for compounds of formula I in which R4 represents R8c, reaction of a corresponding compound of formula I in which R4 represents H with a compound of formula IXD,
L1C(R10a)(R10b)R14xe2x80x83xe2x80x83IXD
wherein R14 represents OC(O)R11, NHC(O)OR12 or OC(O)N(H)R12, and L1, R10a, R10b, R11 and R12 are as hereinbefore defined, for example under conditions described hereinbefore (process step (vi));
(ix) for compounds of formula I in which R1 includes a S(O) or a S(O)2 group, oxidation of a corresponding compound of formula I wherein R1 includes a S group, in the presence of an appropriate amount of a suitable oxidising agent (e.g. mCPBA or potassium peroxymonosulfate) and an appropriate organic solvent (e.g. CH2Cl2, methanol, water or mixtures thereof (e.g. methanol/water)).
Compounds of formula II are available using known and/or standard techniques.
For example, compounds of formula II may be prepared by reaction of an aldehyde of formula X, 
wherein R1 and R2 are as hereinbefore defined, with:
(a) a compound of formula XI,
Rxe2x80x3CNxe2x80x83xe2x80x83XI
wherein Rxe2x80x3 represents H or (CH3)3Si, for example at room, or elevated, temperature (e.g. below 100xc2x0 C.) in the presence of a suitable organic solvent (e.g. chloroform or methylene chloride) and, if necessary, in the presence of a suitable base (e.g. TEA) and/or a suitable catalyst system (e.g. benzylammonium chloride or zinc iodide), followed by hydrolysis under conditions that are well known to those skilled in the art (e.g. as described hereinafter);
(b) NaCN or KCN, for example in the presence of NaHSO3 and water, followed by hydrolysis;
(c) chloroform, for example at elevated temperature (e.g. above room temperature but below 100xc2x0 C.) in the presence of a suitable organic solvent (e.g. chloroform) and, if necessary, in the presence of a suitable catalyst system (e.g. benzylammonium chloride), followed by hydrolysis;
(d) a compound of formula XII, 
wherein M represents Mg or Li, followed by oxidative cleavage (e.g. ozonolysis or osmium or ruthenium catalysed) under conditions which are well known to those skilled in the art; or
(e) tris(methylthio)methane under conditions which are well known to those skilled in the art, followed by hydrolysis in the presence of e.g. HgO and HBF4.
The enantiomeric forms of compounds of formula II (i.e. those compounds having different configurations of substituents about the C-atom xcex1- to the CO2H group) may be separated by an enantiospecific derivatisation step. This may be achieved, for example by an enzymatic process. Such enzymatic processes include, for example, transesterification of the xcex1-OH group at between room and reflux temperature (e.g. at between 45 and 55xc2x0 C.) in the presence of a suitable enzyme (e.g. Lipase PS Amano), an appropriate ester (e.g. vinyl acetate) and a suitable solvent (e.g. methyl tert-butyl ether). The derivatised isomer may then be separated from the unreacted isomer by conventional separation techniques (e.g. chromatography).
Groups added to compounds of formula II in such a derivatisation step may be removed either before any further reactions or at any later stage in the synthesis of compounds of formula I. The additional groups may be removed using conventional techniques (e.g. for esters of the xcex1-OH group, hydrolysis under conditions known to those skilled in the art (e.g. at between room and reflux temperature in the presence of a suitable base (e.g. NaOH) and an appropriate solvent (e.g. MeOH, water or mixtures thereof))).
Compounds of formula III may be prepared by reaction of a compound of formula XIII 
wherein Y is as hereinbefore defined with a compound of formula V as hereinbefore defined, for example under conditions such as those described hereinbefore for synthesis of compounds of formula I (see, for example, process steps (i) and (ii)).
Compounds of formula IV are readily available using known techniques. For example, compounds of formula IV may be prepared by reaction of a compound of formula II as hereinbefore defined with a compound of formula XIII as hereinbefore defined, for example under conditions such as those described hereinbefore for synthesis of compounds of formula I (see, for example, process steps (i) and (ii)).
Compounds of formula V are known in the literature, and/or may be prepared using known techniques. For example compounds of formula V may be prepared by reduction of a compound of formula XIV, 
wherein R3 and R4 are as hereinbefore defined, under conditions that are well known to those skilled in the art.
Compounds of formula VI may be prepared in accordance with peptide coupling techniques, for example in analogous fashion to the methods described hereinbefore for compounds of formula I (see, for example, process steps (i) and (ii)). If desired, compounds of formula VIII may also be prepared in this way.
Compounds of formula IXB may be prepared by reaction of a corresponding compound of formula I in which R4 represents H with an excess of a compound of formula IVA,
R10aC(O)R10bxe2x80x83xe2x80x83XIVA
wherein R10a and R10b are as hereinbefore defined, for example under conditions known to those skilled in the art.
Compounds of formula X are commercially available, are well known in the literature, or are available using known and/or standard techniques.
For example, compounds of formula X may be prepared by reduction of a compound of formula XV, 
wherein R1 and R2 are as hereinbefore defined, in the presence of a suitable reducing agent (e.g. DIBAL-H).
Alternatively, compounds of formula X may be prepared by oxidation of a compound of formula XVI, 
wherein R1 and R2 are as hereinbefore defined, in the presence of a suitable oxidising agent (e.g. pyridinium chlorochromate or a combination of DMSO and oxalyl chloride).
Compounds of formulae II, IV, VI, VIII, X, XV and XVI in which R1 includes a S(O) or a S(O)2 group, may be prepared by oxidation of a corresponding compound of formula II, IV, VI, VIII, X, XV or XVI (as appropriate) wherein R1 includes a S group, for example as described hereinbefore.
Compounds of formulae VII, IX, IXA, IXC, IXD, XI, XII, XIII, XIV, XIVA, XV and XVI, and derivatives thereof, are either commercially available, are known in the literature, or may be obtained either by analogy with the processes described herein, or by conventional synthetic procedures, in accordance with standard techniques, from readily available starting materials using appropriate reagents and reaction conditions (e.g. as described hereinafter).
Substituents on the aromatic and/or non-aromatic, carbocyclic and heterocyclic ring(s) in compounds of formulae I, II, III, IV, V, VI, VII, VIII, IX, IXA, IXB, IXC, IXD, X, XIII, XIV, XV and XVI may be introduced and/or interconverted using techniques well known to those skilled in the art. For example, nitro may be reduced to amino, amino may be alkylated or acylated to give alkyl- and/or acylamino, amino may be converted to pyrrolo (by condensation with a 2,5-dimethoxytetrahydrofuran in the presence of a catalyst, such as phosphorous pentoxide), amino may be converted (via diazotisation) to halo or (e.g. via reaction with a 1,4- or 1,5-dihaloalkyl compound or a xcex2- or xcex3-haloester) to a nitrogen-containing ring (optionally substituted with a xe2x95x90O group), iodo may be converted to nitrogen-containing heterocycles (for example imidazolyl and piperidinyl, by treatment with imidazole or piperidine under Buchwald conditions), nitrogen hydroxy may be alkylated to give alkoxy, alkoxy may be hydrolysed to hydroxy, alkenes may be hydrogenated to alkanes, halo may be hydrogenated to H, etc. In this regard, compounds of formula XV in which R1 represents xe2x80x94N(CH3)2 and R2 represent chloro or methyl, may be obtained from commercially available iodo-chloro or iodo-methyl disubstituted benzoic acid methyl esters using Pd-catalysed amination, for example as described by Wolfe et al in Tetrahedron Lett. 38, 6367 (1997), followed by either reductive amination (for example using HCHO and a reducing agent such as Na(CN)BH3 or a combination of Pt(IV) oxide and hydrogen), or alkylation (for example using MeI and an appropriate base), of the resultant aniline. Compounds of formula XV in which R1 represents xe2x80x94S(O)mCH3 (in which m is as hereinbefore defined) and R2 represents chloro or methyl, may be obtained from the resultant aniline described above (or from the corresponding benzoic acid) via diazotisation, followed by treatment of the diazonium salt with potassium ethyl xanthate, and then hydrolysis of the intermediate to give the corresponding thiophenol, for example as described by Tarbell et al in xe2x80x9cOrganic Synthesisxe2x80x9d, Coll. Vol. III, p 809-11 (1955). The resultant thiophenol may then be alkylated (for example using an appropriate alkyl iodide in the presence of a suitable base in EtOH), and then (if necessary) oxidised to form the sulfone or sulfoxide (for example using mCPBA in CH2Cl2 or potassium peroxymonosulfate in methanouwater).
The compounds of formula I may be isolated from their reaction mixtures using conventional techniques.
It will be appreciated by those skilled in the art that in the processes described above the functional groups of intermediate compounds may need to be protected by protecting groups.
Functional groups which it is desirable to protect include hydroxy, amino, aldehyde, 2-hydroxycarboxylic acid and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl groups (e.g. t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl) and tetrahydropyranyl. Suitable protecting groups for carboxylic acid include C1-6 alkyl or benzyl esters. Suitable protecting groups for amino and amidino include t-butyloxycarbonyl, benzyloxycarbonyl or 2-trimethylsilylethoxycarbonyl (Teoc). Amidino nitrogens may also be protected by hydroxy or alkoxy groups, and may be either mono- or diprotected. Aldehydes may be protected as acetals by reacting with e.g. ethylene glycol. 2-Hydroxy carboxylic acids may be protected by condensing with e.g. acetone.
The protection and deprotection of functional groups may take place before or after coupling, or before or after any other reaction in the abovementioned schemes.
Protecting groups may be removed in accordance with techniques which are well known to those skilled in the art and as described hereinafter.
Persons skilled in the art will appreciate that, in order to obtain compounds of formula I in an alternative, and, on some occasions, more convenient, manner, the individual process steps mentioned hereinbefore may be performed in a different order, and/or the individual reactions may be performed at a different stage in the overall route (i.e. substituents may be added to and/or chemical transformations performed upon, different intermediates to those mentioned hereinbefore in conjunction with a particular reaction). This may negate, or render necessary, the need for protecting groups.
For example, this is particularly true in respect of the synthesis of compounds of formula I in which R4 does not represent H. In this case, OH, OR8a, C(O)OR8b and/or R8c groups may be introduced at an earlier stage in the overall synthesis using the process steps described hereinbefore (see, for example, process steps (iii) to (viii)). Further, the mandelic acid OH group of compounds of formulae II and IV may need to be protected prior to the coupling steps described above.
Accordingly, the order and type of chemistry involved will dictate the need, and type, of protecting groups as well as the sequence for accomplishing the synthesis.
The use of protecting groups is fully described in xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d, edited by J W F McOmie, Plenum Press (1973), and xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d, 2nd edition, T W Greene and P G M Wutz, Wiley-Interscience (1991).
Protected derivatives of compounds of formula I may be converted chemically to compounds of formula I using standard deprotection techniques (e.g. hydrogenation). The skilled person will also appreciate that certain compounds of formula I may also be referred to as being xe2x80x9cprotected derivativesxe2x80x9d of other compounds of formula I.
Compounds of the invention may possess pharmacological activity as such. Compounds of the invention that may possess such activity include, but are not limited to, those in which R4 is H.
However, other compounds of formula I (including those in which R4 is not H) may not possess such activity, but may be administered parenterally or orally, and thereafter metabolised in the body to form compounds that are pharmacologically active (including, but not limited to, corresponding compounds in which R4 is H). Such compounds (which also includes compounds that may possess some pharmacological activity, but that activity is appreciably lower than that of the xe2x80x9cactivexe2x80x9d compounds to which they are metabolised), may therefore be described as xe2x80x9cprodrugsxe2x80x9d of the active compounds.
Thus, the compounds of the invention are useful because they possess pharmacological activity, and/or are metabolised in the body following oral or parenteral administration to form compounds which possess pharmacological activity. The compounds of the invention are therefore indicated as pharmaceuticals.
According to a further aspect of the invention there is thus provided the compounds of the invention for use as pharmaceuticals.
In particular, compounds of the invention are potent inhibitors of thrombin either as such and/or (e.g. in the case of prodrugs), are metabolised following administration to form potent inhibitors of thrombin, for example as demonstrated in the tests described below.
By xe2x80x9cprodrug of a thrombin inhibitorxe2x80x9d, we include compounds that form a thrombin inhibitor, in an experimentally-detectable amount, and within a predetermined time (e.g. about 1 hour), following oral or parenteral administration.
The compounds of the invention are thus expected to be useful in those conditions where inhibition of thrombin is required.
The compounds of the invention are thus indicated in the treatment and/or prophylaxis of thrombosis and hypercoagulability in blood and tissues of animals including man.
It is known that hypercoagulability may lead to thrombo-embolic diseases. Conditions associated with hypercoagulability and thrombo-embolic diseases which may be mentioned include inherited or acquired activated protein C resistance, such as the factor V-mutation (factor V Leiden), and inherited or acquired deficiencies in antithrombin III, protein C, protein S or heparin cofactor II. Other conditions known to be associated with hypercoagulability and thrombo-embolic disease include circulating antiphospholipid antibodies (Lupus anticoagulant), homocysteinemi, heparin induced thrombocytopenia and defects in fibrinolysis. The compounds of the invention are thus indicated both in the therapeutic and/or prophylactic treatment of these conditions.
The compounds of the invention are further indicated in the treatment of conditions where there is an undesirable excess of thrombin without signs of hypercoagulability, for example in neurodegenerative diseases such as Alzheimer""s disease.
Particular disease states which may be mentioned include the therapeutic and/or prophylactic treatment of venous thrombosis and pulmonary embolism, arterial thrombosis (e.g. in myocardial infarction, unstable angina, thrombosis-based stroke and peripheral arterial thrombosis) and systemic embolism usually from the atrium during arterial fibrillation or from the left ventricle after transmural myocardial infarction.
Moreover, the compounds of the invention are expected to have utility in prophylaxis of re-occlusion (i.e. thrombosis) after thrombolysis, percutaneous trans-luminal angioplasty (PTA) and coronary bypass operations; the prevention of re-thrombosis after microsurgery and vascular surgery in general.
Further indications include the therapeutic and/or prophylactic treatment of disseminated intravascular coagulation caused by bacteria, multiple trauma, intoxication or any other mechanism; anticoagulant treatment when blood is in contact with foreign surfaces in the body such as vascular grafts, vascular stents, vascular catheters, mechanical and biological prosthetic valves or any other medical device; and anticoagulant treatment when blood is in contact with medical devices outside the body such as during cardiovascular surgery using a heart-lung machine or in haemodialysis.
In addition to its effects on the coagulation process, thrombin is known to activate a large number of cells (such as neutrophils, fibroblasts, endothelial cells and smooth muscle cells). Therefore, the compounds of the invention may also be useful for the therapeutic and/or prophylactic treatment of idiopathic and adult respiratory distress syndrome, pulmonary fibrosis following treatment with radiation or chemotherapy, septic shock, septicemia, inflammatory responses, which include, but are not limited to, edema, acute or chronic atherosclerosis such as coronary arterial disease, cerebral arterial disease, peripheral arterial disease, reperfusion damage, and restenosis after percutaneous trans-luminal angioplasty (PTA).
Compounds of the invention that inhibit trypsin and/or thrombin may also be useful in the treatment of pancreatitis.
According to a further aspect of the present invention, there is provided a method of treatment of a condition where inhibition of thrombin is required which method comprises administration of a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, to a person suffering from, or susceptible to such a condition.
The compounds of the invention will normally be administered orally, intravenously, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, by any other parenteral route or via inhalation, in the form of pharmaceutical preparations comprising active compound either as a free base, or a pharmaceutically acceptable non-toxic organic or inorganic acid addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated and the route of administration, the compositions may be administered at varying doses.
The compounds of the invention may also be combined and/or co-administered with any antithrombotic agent with a different mechanism of action, such as the antiplatelet agents acetylsalicylic acid, ticlopidine, clopidogrel, thromboxane receptor and/or synthetase inhibitors, fibrinogen receptor antagonists, prostacyclin mimetics and phosphodiesterase inhibitors and ADP-receptor (P2T) antagonists.
The compounds of the invention may further be combined and/or co-administered with thrombolytics such as tissue plasminogen activator (natural, recombinant or modified), streptokinase, urokinase, prourokinase, anisoylated plasminogen-streptokinase activator complex (APSAC), animal salivary gland plasminogen activators, and the like, in the treatment of thrombotic diseases, in particular myocardial infarction.
According to a further aspect of the invention there is thus provided a pharmaceutical formulation including a compound of the invention, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.
Suitable daily doses of the compounds of the invention in therapeutical treatment of humans are about 0.001-100 mg/kg body weight at peroral administration and 0.001-50 mg/kg body weight at parenteral administration.
The compounds of the invention have the advantage that they may, or may be metabolised to compounds that may, be more efficacious, be less toxic, be longer acting, have a broader range of activity, be more potent, produce fewer side effects, be more easily absorbed than, or have other useful pharmacological, physical, or chemical, properties over, compounds known in the prior art.
The inhibitor solution (25 xcexcL) was incubated with plasma (25 xcexcL) for three minutes. Human thrombin (T 6769; Sigma Chem. Co or Hematologic Technologies) in buffer solution, pH 7.4 (25 xcexcL, 4.0 NIH units/mL), was then added and the clotting time measured in an automatic device (KC 10; Amelung).
The thrombin clotting time (TT) is expressed as absolute values (seconds) as well as the ratio of TT without inhibitor (TT0) to TT with inhibitor (TTi). The latter ratios (range 1-0) were plotted against the concentration of inhibitor (log transformed) and fitted to sigmoidal dose-response curves according to the equation
y=a/[1+(x/IC50)S]
where: a=maximum range, i.e. 1; s=slope of the dose-response curve; and IC50=the concentration of inhibitor that doubles the clotting time. The calculations were processed on a PC using the software program GraFit Version 3, setting equation equal to: Start at 0, define end=1 (Erithacus Software, Robin Leatherbarrow, Imperial College of Science, London, UK).
Determination of Thrombin Inhibition with a Chromogenic, Robotic Assay
The thrombin inhibitor potency was measured with a chromogenic substrate method, in a Plato 3300 robotic microplate processor (Rosys AG, CH-8634 Hombrechtikon, Switzerland), using 96-well, half volume microtitre plates (Costar, Cambridge, Mass., USA; Cat No 3690). Stock solutions of test substance in DMSO (72 xcexcL), 0.1-1 mmol/L, were diluted serially 1:3 (24+48 xcexcL) with DMSO to obtain ten different concentrations, which were analysed as samples in the assay. 2 xcexcL of test sample was diluted with 124 xcexcL assay buffer, 12 xcexcL of chromogenic substrate solution (S-2366, Chromogenix, Mxc3x6lndal, Sweden) in assay buffer and finally 12 xcexcL of xcex1-thrombin solution (Human xcex1-thrombin, Sigma Chemical Co. or Hematologic Technologies) in assay buffer, were added, and the samples mixed. The final assay concentrations were: test substance 0.00068-13.3 xcexcmol/L, S-2366 0.30 mmol/L, xcex1-thrombin 0.020 NIHU/mL. The linear absorbance increment during 40 minutes incubation at 37xc2x0 C. was used for calculation of percentage inhibition for the test samples, as compared to blanks without inhibitor. The IC50-robotic value, corresponding to the inhibitor concentration which caused 50% inhibition of the thrombin activity, was calculated from a log concentration vs. % inhibition curve.
Determination of the Inhibition Constant K for Human Thrombin Ki-determinations were made using a chromogenic substrate method, performed at 37xc2x0 C. on a Cobas Bio centrifugal analyser (Roche, Basel, Switzerland). Residual enzyme activity after incubation of human xcex1-thrombin with various concentrations of test compound was determined at three different substrate concentrations, and was measured as the change in optical absorbance at 405 nm.
Test compound solutions (100 xcexcL; normally in buffer or saline containing BSA 10 g/L) were mixed with 200 xcexcL of human xcex1-thrombin (Sigma Chemical Co) in assay buffer (0.05 mol/L Tris-HCl pH 7.4, ionic strength 0.15 adjusted with NaCl) containing BSA (10 g/L), and analysed as samples in the Cobas Bio. A 60 xcexcL sample, together with 20 xcexcL of water, was added to 320 xcexcL of the substrate S-2238 (Chromogenix AB, Mxc3x6lndal, Sweden) in assay buffer, and the absorbance change (xcex94A/min) was monitored. The final concentrations of S-2238 were 16, 24 and 50 xcexcmol/L and of thrombin 0.125 NIH U/mL.
The steady state reaction rate was used to construct Dixon plots, i.e. diagrams of inhibitor concentration vs. 1/(xcex94A/min). For reversible, competitive inhibitors, the data points for the different substrate concentrations typically form straight lines which intercept at x=-Ki.
Determination of Activated Partial Thromboplastin Time (APTT)
APTT was determined in pooled normal human citrated plasma with the reagent PTT Automated 5 manufactured by Stago. The inhibitors were added to the plasma (10 xcexcL inhibitor solution to 90 xcexcL plasma) and incubated with the APTT reagent for 3 minutes followed by the addition of 100 xcexcL of calcium chloride solution (0.025 M) and APTT was determined by use of the coagulation analyser KC10 (Amelung) according to the instructions of the reagent producer.
The clotting time is expressed as absolute values (seconds) as well as the ratio of APTT without inhibitor (APTT0) to APTT with inhibitor (APTTi). The latter ratios (range 1-0) were plotted against the concentration of inhibitor (log transformed) and fitted to sigmoidal dose-response curves according to the equation
y=a/[1+(x/IC50)S]
where: a=maximum range, i.e. 1; s=slope of the dose-response curve; and IC50=the concentration of inhibitor that doubles the clotting time. The calculations were processed on a PC using the software program GraFit Version 3, setting equation equal to: Start at 0, define end=1 (Erithacus Software, Robin Leatherbarrow, Imperial College of Science, London, UK).
IC50APTT is defined as the concentration of inhibitor in human plasma that doubled the Activated Partial Thromboplastin Time.
Determination of Thrombin Time Ex Vivo
The inhibition of thrombin after oral or parenteral administration of the compounds of formula I, dissolved in ethanol:Solutol(trademark):water (5:5:90), were examined in conscious rats which, one or two days prior to the experiment, were equipped with a catheter for blood sampling from the carotid artery. On the experimental day blood samples were withdrawn at fixed times after the administration of the compound into plastic tubes containing 1 part sodium citrate solution (0.13 mol per L) and 9 parts of blood. The tubes were centrifuged to obtain platelet poor plasma. The plasma was used for determination of thrombin time or ecarin clotting time (ECT) as described below.
The citrated rat plasma, 100 xcexcL, was diluted with a saline solution, 0.9%, 100 xcexcL, and plasma coagulation was started by the addition of human thrombin (T 6769, Sigma Chem Co, USA or Hematologic Technologies) in a buffer solution, pH 7.4, 100 xcexcL, or ecarin (Pentapharm). The clotting time was measured in an automatic device (KC 10, Amelung, Germany).
Where a xe2x80x9cprodrugxe2x80x9d compound of formula I was administered, concentrations of the appropriate active thrombin inhibitor of formula I (e.g. the free amidine compound) in the rat plasma were estimated by the use of standard curves relating the thrombin time or ecarin clotting time in the pooled citrated rat plasma to known concentrations of the corresponding xe2x80x9cactivexe2x80x9d thrombin inhibitor dissolved in saline.
Based on the estimated plasma concentrations of the active thrombin inhibitor (which assumes that thrombin time or ECT prolongation is caused by the aforementioned compound) in the rat, the area under the curve after oral and/or parenteral administration of the corresponding prodrug compound of formula I was calculated (AUCpd) using the trapezoidal rule and extrapolation of data to infinity.
The bioavailability of the active thrombin inhibitor after oral or parenteral administration of the prodrug was calculated as below:
xe2x80x83[(AUCpd/dose)/(AUCactive,parenteral/dose]xc3x97100
where AUCactive,parenteral represents the AUC obtained after parenteral administration of the corresponding active thrombin inhibitor to conscious rats as described above.
Determination of Thrombin Time in Urine Ex Vivo
The amount of the xe2x80x9cactivexe2x80x9d thrombin inhibitor that was excreted in urine after oral or parenteral administration of xe2x80x9cprodrugxe2x80x9d compounds of the invention, dissolved in ethanol:Solutol(trademark):water (5:5:90), was estimated by determination of the thrombin time in urine ex vivo (assuming that thrombin time prolongation is caused by the aforementioned compound).
Conscious rats were placed in metabolism cages, allowing separate collection of urine and faeces, for 24 hours following oral administration of compounds of the invention. The thrombin time was determined on the collected urine as described below.
Pooled normal citrated human plasma (100 xcexcL) was incubated with the concentrated rat urine, or saline dilutions thereof, for one minute. Plasma coagulation was then initiated by the administration of human thrombin (T 6769, Sigma Chem Company) in buffer solution (pH 7.4; 100 xcexcL). The clotting time was measured in an automatic device (KC 10; Amelung).
The concentrations of the active thrombin inhibitor in the rat urine were estimated by the use of standard curves relating the thrombin time in the pooled normal citrated human plasma to known concentrations of the aforementioned active thrombin inhibitor dissolved in concentrated rat urine (or saline dilutions thereof). By multiplying the total rat urine production over the 24 hour period with the estimated mean concentration of the aforementioned active inhibitor in the urine, the amount of the active inhibitor excreted in the urine (AMOUNTpd) could be calculated.
The bioavailability of the active thrombin inhibitor after oral or parenteral administration of the prodrug was calculated as below:
[(AMOUNTpd/dose)/(AMOUNTactive,parenteral/dose]xc3x97100
where AMOUNTactive,parenteral represents the amount excreted in the urine after parenteral administration of the corresponding active thrombin inhibitor to conscious rats as described above.
Metabolic Activation of Prodrug Compounds In Vitro
Prodrug compounds of formula I were incubated at 37xc2x0 C. with liver microsomes or 10,000 g (referring to the centrifuge speed) supernatant fractions (i.e. s9 fraction) prepared from human or rat liver homogenate. The total protein concentration in the incubations were 1 or 3 mg/mL dissolved in 0.05 mol/L TRIS buffer (pH 7.4), and with the cofactors NADH (2.5 mmol/L) and NADPH (0.8 mmol/L) present. The total volume of the incubate was 1.2 mL. The initial prodrug concentrations were 5 or 10 xcexcmol/L. Samples were collected from the incubate at regular intervals more than 60 minutes after the start of the incubations. Samples (25 xcexcL) from the incubate were mixed with an equal volume of human or rat plasma and an appropriate amount of thrombin, and the clotting time (i.e. thrombin time) was measured on a coagulometer (KC 10; Amelung). The amount of xe2x80x9cactivexe2x80x9d thrombin inhibitor formed was estimated by the use of standard curves relating the thrombin time in pooled citrated human or rat plasma to known concentrations of the corresponding xe2x80x9cactive thrombin inhibitorxe2x80x9d.
The amount of xe2x80x9cactivexe2x80x9d thrombin inhibitor was alternatively, or in addition to the above-mentioned method, estimated by the use of LC-MS.